Foehrenbacher Optimizedhypoxiaactivatedprodrugs Pharmacokineticmodeling 2013

HYPOTHESIS AND THEORY ARTICLE
published: 27 December 2013

doi: 10.3389/fonc.2013.00314
Design of optimized hypoxia-activated prodrugs using

pharmacokinetic/pharmacodynamic modeling
Annika Foehrenbacher 1 , Timothy W. Secomb 2 , William R. Wilson 1 and Kevin O. Hicks 1 *
1
2
Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand
Department of Physiology, University of Arizona, Tucson, AZ, USA
Edited by:
Annarosa Arcangeli, University of
Florence, Italy
Reviewed by:
Carine Michiels, University of Namur,
Belgium
Brion William Murray, Pfizer Oncology
Research Unit, USA
*Correspondence:
Kevin O. Hicks, Experimental
Therapeutics Group, Auckland Cancer
Society Research Centre, The
University of Auckland, 85 Park Road,
Auckland 1142, New Zealand
e-mail: k.hicks@auckland.ac.nz
Hypoxia contributes to resistance of tumors to some cytotoxic drugs and to radiotherapy,

but can in principle be exploited with hypoxia-activated prodrugs (HAP). HAP in clinical
development fall into two broad groups. Class I HAP (like the benzotriazine N-oxides tirapazamine and SN30000), are activated under relatively mild hypoxia. In contrast, Class II
HAP (such as the nitro compounds PR-104A or TH-302) are maximally activated only under
extreme hypoxia, but their active metabolites (effectors) diffuse to cells at intermediate O2
and thus also eliminate moderately hypoxic cells. Here, we use a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model to compare these two strategies and to
identify the features required in an optimal Class II HAP. The model uses a Greens function
approach to calculate spatial and longitudinal gradients of O2 , prodrug, and effector concentrations, and resulting killing in a digitized 3D tumor microregion to estimate activity as
monotherapy and in combination with radiotherapy. An analogous model for a normal tissue
with mild hypoxia and short intervessel distances (based on a cremaster muscle microvessel network) was used to estimate tumor selectivity of cell killing. This showed that Class II
HAP offer advantages over Class I including higher tumor selectivity and greater freedom
to vary prodrug diffusibility and rate of metabolic activation. The model suggests that the
largest gains in class II HAP antitumor activity could be realized by optimizing effector stability and prodrug activation rates. We also use the model to show that diffusion of effector
into blood vessels is unlikely to materially increase systemic exposure for realistic tumor
burdens and effector clearances. However, we show that the tumor selectivity achievable
by hypoxia-dependent prodrug activation alone is limited if dose-limiting normal tissues are
even mildly hypoxic.
Keywords: tumor hypoxia, hypoxia-activated prodrugs, bystander effect, extravascular drug transport,
pharmacokinetic/pharmacodynamic modeling, rational drug design, tirapazamine, PR-104
INTRODUCTION
Prodrugs that are enzymatically converted to active metabolites
(effectors) within tumors are of interest for selective cancer therapy. Three different strategies have been explored for intra-tumor
prodrug activation: (1) xenobiotic metabolizing enzymes that are
highly expressed in specific tumors (1, 2); (2) exogenous enzymes
targeted to tumors using antibody (3), viral (4), or bacterial (5)
vectors; (3) endogenous enzymes that reduce prodrugs only under
the hypoxic conditions that prevail in tumors. Hypoxia-activated
prodrugs (HAP), which provide this third strategy, not only exploit
a generic feature that differentiates tumors from most normal tissues but also potentially overcome the resistance of hypoxic cells to
radiotherapy and many chemotherapy drugs (615). Several HAP
have been evaluated clinically (1619), including ongoing studies
with the nitro compounds TH-302 (20, 21), PR-104A [(2225);
administered as the corresponding phosphate ester PR-104] and
PR-610 (26). However, many questions remain as to how HAP
should be designed for optimal anticancer activity.
In the present study we utilize pharmacokinetic/
pharmacodynamic (PK/PD) models to explore relationships
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between the reaction/diffusion properties of HAP in the tumor

microenvironment and their antitumor activity and selectivity.
In this context it is useful to distinguish two broad classes of HAP
with different PK/PD features (Figure 1). Class I HAP are activated
by reduction under relatively mild hypoxia to generate a reactive
cytotoxin, which is restricted to the cell in which it is formed. This
class is typified by the benzotriazine N -oxides tirapazamine and
SN30000 that undergo 1-electron reduction to short-lived cytotoxic radicals (27, 28), which appear not to affect adjoining cells
in three-dimensional (3D) cell cultures (29). The O2 concentration for 50% inhibition of cytotoxicity (KO2 ) in stirred single cell
suspensions is approximately 1 M for both tirapazamine (30, 31)
and SN30000 (32). Class II HAP (typified by nitro compounds
such as PR-104A and TH-302) require more severe hypoxia for 1electron reduction, but the resulting radicals are further reduced
to a relatively stable effector that can diffuse from the cell of origin
(29, 3335); this diffusion of effector to nearby cells (which may
not themselves be capable of prodrug activation) is here referred
to as a bystander effect. In the case of PR-104A, a KO2 value of
~0.1 M has been estimated in stirred single cell suspensions (31),
December 2013 | Volume 3 | Article 314 | 1
Foehrenbacher et al.
FIGURE 1 | Hypoxia-activated prodrug strategies. (A) Schematic

representation of complementary cell killing achieved by radiation in
combination with a high-KO2 HAP and a low-KO2 HAP that generates active
metabolites that diffuse out of prodrug-activating zones to kill neighboring
cells (bystander). (B) Examples of Class I (SN30000) and Class II (PR-104A)
HAP are shown.
and activation of TH-302 also appears to require severe hypoxia

(34) as is the case for other nitro compounds (3638). Given that
half-maximal radiosensitization of tumor cells by oxygen requires
concentrations of ~4 M (39, 40), the ability of Class II HAP to
overcome radioresistance may depend on bystander effects.
We have suggested (41) that Class II HAP may be preferable to
Class I because activation will be confined to the extreme hypoxia
found in tumors, thus minimizing toxicity to normal tissues with
mild, physiological hypoxia [e.g., retina (42), liver (4345), esophagus (46), skin (47, 48), and possibly the bone marrow stem cell
niche (49) although the oxygenation status of the latter is controversial (50)]. The bystander effect from Class II HAP may
contribute to the reported monotherapy activity of PR-104 (33,
51, 52) and TH-302 (53) in preclinical models. However, it is not
known under what conditions this theoretical advantage for Class
II HAP might be realized, or how this activity might be optimized
by prodrug design, or whether diffusion of active metabolites into
the tumor microvasculature might contribute to systemic toxicity
if this process is too efficient.
Spatially resolved pharmacokinetic/pharmacodynamic (SRPK/PD) models provide tools for addressing these questions.
These models can be used to describe concentration gradients
of oxygen, HAP, and their effectors in tumors, using mapped
microvascular networks, and to calculate resulting reproductive
cell death (clonogenic cell killing). These models include the
effects of heterogeneity in inter-capillary distances, vessel diameters, blood flow rates, and vessel oxygen and drug concentrations.
We have validated an SR-PK/PD model for Class I HAP by showing that it predicts activity of tirapazamine analogs combined with
radiotherapy in human tumor xenografts (32, 54, 55). This modeling clearly demonstrated the need to optimize rates of reductive
metabolism such that penetration into hypoxic regions is not compromised by excessive consumption of the prodrug. Recently, we
have also reported an SR-PK/PD model for the Class II HAP,
PR-104, and used this to estimate that 3050% of its activity in
Frontiers in Oncology | Pharmacology of Anti-Cancer Drugs
Model-based optimization of hypoxia-activated prodrugs
FIGURE 2 | Schematic representation of a generalized HAP PK/PD

model. Transfer of prodrug and effector between the extracellular and the
intracellular compartment is defined by rate constant k t N , where N refers to
each compound. In the extracellular compartment compounds can diffuse
as defined by their diffusion coefficients DN (double-headed arrows).
Prodrug activation (defined by k met P ) is restricted to the intracellular
compartment and is O2 -dependent (see Eq. 3). For an effector that confers
cytotoxicity by way of reacting irreversibly with its target (case 1, e.g.,
DNA-alkylating agents) the model assumes that potency a scales with
reactivity of the effector (k met E ), while in case 2 (e.g., a reversible inhibitor) a
is independent of k met E .
HCT116 and SiHa xenografts is due to bystander effects both as

monotherapy and combined with radiation (35). Here, we use
a generalized SR-PK/PD model, in which a HAP is metabolized
by an oxygen-inhibited process to a single effector (Figure 2), to
ask under what conditions Class II HAP might provide greater
tumor activity and selectivity than Class I HAP, and to identify
the prodrug features required for optimal antitumor activity. This
generalized HAP model makes explicit the diffusion of both the
prodrug and effector in the extracellular (interstitial) compartment. We consider two types of effector, which elicit cytotoxicity
via irreversible reaction with a target (Case 1, as for an alkylating
agent) or by reversible binding to its target (Case 2, as for a receptor
ligand).
METHODS
The generalized SR-PK/PD model calculates steady-state concentrations of oxygen, HAP, and effector as well as resulting cell killing
in digitized 3D tissue microregions using Greens function methods (35, 54, 56). We used two different tissue microregions that
were derived by mapping microvascular anatomy as well as direction and velocity of blood flow in a rat cremaster muscle (56)
(normal network) and a subcutaneous FaDu tumor xenograft
(57) (tumor network). The blood vessels are represented by
cylindrical segments and vessel walls are treated as part of the tissue
space. The model was implemented using a customized version of
the Greens function method written in Visual C++ (Microsoft
Visual Studio 2010 Express) (35, 58).
CALCULATION OF OXYGENATION
Convective transport of oxygen along vessel segments and diffusion into the surrounding tissue (represented as homogeneous
medium) was calculated based on previous estimates for blood

flow in the networks, blood O2 content, tissue diffusion, and
consumption of O2 (35, 56). All O2 transport parameters are
summarized in Table 1.
production equal to the rate of loss of prodrug: rE = i k metP C iP .

The rate constant for prodrug activation is O2 -dependent and is
given by:

kmetP =
CALCULATION OF PHARMACOKINETICS
Inflow of prodrug to the microvascular networks was defined by its

unbound plasma level, using area under the concentration-time
curve (AUC) as a time-independent exposure variable compatible with Greens function formalism. Vessel walls were modeled as offering negligible resistance to radial flux by setting the
intravascular resistance constant K (56) to a low value (0.1 s/m).
Extravascular transport was calculated using a (pro)drug transport model (Figure 2) assuming that the tissue consists of
two homogeneous (extracellular and intracellular) compartments,
with activation of prodrug restricted to the intracellular compartment and diffusion confined to the extracellular compartment. Concentrations in the two compartments were calculated
as follows:
Ce N
e
= DN 2 CeN i ktN (CeN CiN )
t
CiN
i
= i ktN (CeN CiN ) i kmetN CiN + rN
t
(1)
(2)
C e and C i are the extracellular and intracellular concentrations, respectively, of prodrug or effector (denoted by N = P or
E), i and e are the intra- and extracellular volume fractions
with e = 1 i , k tN are first order rate constants for cell transfer
between the extracellular and intracellular compartments, DN is
the effective diffusion coefficient of compound N, 52 is the Laplacian operator, k metN is the rate constant for drug metabolism and r
is the rate of metabolic production with rP = 0 and rate of effector

KO 2
kmetP,max
KO 2 + [O2 ]
(3)
where k metP,max and k metP are the prodrug activation rate constants under anoxia and at the O2 concentration [O2 ], respectively,
and KO2 is the O2 concentration for half-maximum prodrug
activation.
CALCULATION OF CELL KILLING
Survival probability (SP) at each point of the tumor microregion

was calculated using the PK/PD relationship:
log Survival Probability (SP) = aAU CiE
(4)
where AUCiE is the intracellular exposure (area under the intracellular concentration-time curve) to the effector and a is a proportionality constant (potency) equal to the reciprocal of the effector
AUC to produce a SP of 0.1 as measured by clonogenic cell survival.
We distinguish two cases: case 1, where potency a scales with
k metE , broadly representing irreversible inhibitors such as DNAalkylating agents for which an increase in reactivity has similar
effects on reaction with target and non-target nucleophiles (59, 60)
and case 2, where a is independent of k metE (broadly representing
reversible inhibitors). Case 1, where cell killing is proportional to
the reactivity of the effector is used throughout this study unless
noted otherwise.
To estimate cell SP after radiation treatment, the linearquadratic model was used as previously (35, 54). The SP from
Table 1 | O2 transport parameters used to model oxygenation in the FaDu tumor and the cremaster muscle microregions.
Parameter
Unit
Value for
FaDu
Description
Cremaster
mm3
0.12
0.091
Volume of the mapped region
nl/min
40.0
75.0
Total blood inflow to region = sum of blood flow in all feeding arterioles
Q/V
nl/min/mm3
333
824
pO2
mm Hg
40.0
50.0
O2 partial pressure in inflowing vessels
P 50
mm Hg
38.0
38.0
O2 partial pressure for half saturation of hemoglobin
3.0
3.0
Hill coefficient for O2 binding to hemoglobin
0.5
0.5
O2 binding capacity of red blood cells
N
C0
cm3 O2 /mm Hg
HD
cm3 O2 /cm3 /mm Hg
0.4
0.4
Discharge hematocrit
3.1 105
3.1 105
O2 solubility
Krogh diffusion coefficient for O2 diffusion
DO2
cm3 O2 /cm/s/mm Hg
4.2 1010
9.4 1010
DO2
cm2 /s
1.4 105
3.0 105
O2 diffusion coefficient
M0
cm3 O2 /cm3 /s
5 104
9.5 104
Rate of O2 consumption when supply is not limiting
KmO2
mm Hg
1.0
1.0
Michaelis constant for O2 consumption
Parameters were as reported previously for the FaDu (35) and the cremaster muscle microregion (56) except that M0 in the cremaster region was adjusted to achieve
the required hypoxic fraction. The chosen M0 value is intermediate between the values previously used to represent mild and moderate levels of muscle activity (56).
These O2 transport parameters gave hypoxic fractions and median pO2 values (Figure 3) in the range measured for tumors and normal tissues in humans using O2
electrode histography (45).
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both radiation and prodrug at each point of the tumor microregion was calculated from the sum of log (SP) due to drug and
radiation alone. Log cell kill was then calculated as
log cell kill = log(Average SP)
(5)
where the SP is averaged over the whole tumor microregion.

Prodrug-induced cell kill in addition to radiation was calculated as
the difference between overall log cell kill by prodrug + radiation
and log cell kill by radiation alone.
ESTIMATION OF NET TRANSPORT ACROSS THE TUMOR-PLASMA
BOUNDARY
The SR-PK/PD model calculates the transport of prodrug and

effector across the walls of the 582 vessel segments in the FaDu
tumor microvascular network. Summing the 582 values for each
compound gives the net transport across the tumor-plasma
boundary (in picomoles). This was normalized to the volume of
the FaDu tumor microregion.
COMPOUNDS
PR-104H (33) and PR-104M (61) were prepared from PR-104A

as described and stored in acetonitrile at 80C. Tetradeuterated internal standards of PR-104H (PR-104H-d4) and PR-104M
(PR-104M-d4) were synthesized using the same methods as for
the non-deuterated compounds. PR-104H and PR-104H-d4 had
a purity of >90% by HPLC while PR-104M and PR-104M-d4 had
a purity of 86 and 84%, respectively.
PHARMACOKINETIC STUDIES IN MICE
All animal studies were approved by the University of Auckland Animal Ethics Committee. Male NIH-III nude mice (NIHLystbg Foxn1nu Btkxid ; 1820 g body weight), derived from breeding
mice purchased from Charles River Laboratories (Wilmington,
MA, USA), were dosed i.v. with PR-104H. The dosing solution was
prepared fresh for each mouse by dilution of a stock solution of
PR-104H in acetonitrile with sterile saline (to <20% acetonitrile)
within 3 min of dosing to minimize loss of PR-104H in aqueous solution. At the dose volume of 0.005 ml/g the acetonitrile
dose was 755 mg/kg and the vehicle alone did not cause signs
of toxicity over the duration of the experiment. Blood was collected by cardiac puncture up to 3 min after cervical dislocation
as in (62). Plasma was prepared as described (63) and mixed with
three volumes of cold acidified methanol (methanol:ammonium
acetate:acetic acid 1000:3.5:0.2, v/w/v) ~6 min after cardiac puncture. Liver tissue was dissected within 3 min of blood sampling,
snap-frozen in liquid nitrogen, and stored at 80C along with
plasma samples. Frozen tissue was pulverized at liquid nitrogen
temperature using a BioPulverizer (BioSpec Products, USA),
transferred to tared tubes, weighed, vortex mixed for 30 s with an
equal volume of cold phosphate buffered saline and six volumes
of cold acidified methanol, and stored at 80C. For LC-MS/MS
analysis [as reported in Ref. (35)], tissue and plasma extracts
were centrifuged (13,000 g, 4C, 10 min) and supernatants were
mixed 1:1 with cold water containing 1 M PR-104H-d4 and 1 M
PR-104M-d4.
RESULTS
In order to identify optimal properties of HAP, we developed a
SR-PK/PD model that captures the key features of both Class I
and Class II HAP (Figure 2). This model is a generalized form
of our SR-PK/PD model for PR-104 (35), which, unlike earlier
SR-PK/PD models (31, 32, 54) accommodates diffusion and reaction of the effector and explicitly considers intra- and extracellular
compartments in order to represent the plasma membrane barrier to prodrug uptake and effector efflux. Our generalized model
assumes that the effector can be further converted to inactive products within the cell (defined by rate constant k metE ). The model
incorporates only one effector (unlike the two alkylating metabolites from PR-104A) and represents an idealized HAP that is not
a substrate for O2 -insensitive two-electron-reduction, which is an
off-target activation mechanism of some HAP including PR-104A
(51). We also ignore any systemic generation of effector outside
the modeled tissue region, such as from hepatic metabolism.
TISSUE MICROREGIONS USED FOR SR-PK/PD MODELING
We used a digitized 3D tumor microregion (Figure 3A) that was

derived by mapping a region of a subcutaneous FaDu tumor
xenograft grown in a mouse dorsal window chamber (57). To
assess HAP activation under conditions of physiological hypoxia
we also used a 3D region of a rat cremaster muscle (56) as an
example of a normal tissue with a well-organized and efficient
microvascular network (Figure 3B). While the maximum distance to the nearest blood vessel is 50 m in the cremaster muscle
region, 20% of tissue points in the FaDu tumor region are situated
further from blood vessels (50100 m; Figure 3C). The FaDu
tumor region also showed a higher heterogeneity in vessel diameters (Figure 3D), and a lower median blood flow rate (Figure 3E)
at the values chosen for total blood inflow into the regions (Q;
Table 1). Q per tissue volume was 2.5-fold lower in the FaDu
tumor than in the cremaster muscle microregion, which is realistic for a low-perfused hypoxic tumor area. The value for the tumor
microregion falls within the range of mean blood flow values measured in human tumors, which show high variability with values
that can be lower, similar, or higher than in the tissue of origin
(64, 65). A radiobiological hypoxic fraction (<4 M O2 ) of 43%
was achieved in the FaDu tumor microregion (Figure 3F) by using
O2 transport parameters previously chosen to model the hypoxic
fraction of tumor xenografts (35) (Table 1). The O2 transport
parameters for the cremaster muscle region were as reported (56),
with the oxygen consumption rate adjusted to achieve a radiobiological hypoxic fraction of 7.5% (Figure 3F) that is consistent
with moderate hypoxia as measured in some normal human tissues using O2 electrode histography (45). However, the muscle
region lacks the severely hypoxic cells (defined as <0.13 M O2 ,
i.e., below the KO2 value for PR-104A) that comprise 5.0% of the
FaDu microregion (Figure 3F).
MODULATION OF KINETICS OF PRODRUG ACTIVATION
Our previous SR-PK/PD model for Class I HAP showed that high
prodrug activation rates limit killing of hypoxic cells by impairing tissue penetration of the prodrug (54). To investigate whether
this also applies to HAP with bystander effects and/or activation
restricted to more severe hypoxia (low KO2 ), we modulated the
FIGURE 3 | Virtual tissue microregions used for SR-PK/PD modeling.

Oxygenation in the FaDu tumor microregion [990 m 810 m
150 m; (A)] and the cremaster muscle region [1100 m 1000 m
240 m; (B)] was modeled using the O2 transport parameters reported in
Table 1. (A,B) Show contour plots of O2 (in mm Hg) in a mid-plane
section of the microregions, superimposed with the whole microvascular
network projected into one plane. The dimensions of the mapped
prodrug activation rate constant for anoxia (k metP,max ) under these

conditions. For simulations without a bystander effect, k tE was set
to 0, which traps effector within the intracellular compartment.
Increasing k metP,max resulted in a decrease of prodrug penetration to the most hypoxic regions, an effect that was less pronounced with a low KO2 (Figure 4A) than with a high KO2 value
(Figure 4D), as expected. Accordingly, killing in the most hypoxic
regions (<0.13 M O2 ) was higher and less affected by an increase
in k metP,max for low-KO2 HAP (Figures 4B,E; without bystander
effects). The observed shift of cell killing to higher O2 concentrations with increasing k metP,max reflects increased prodrug
activation at intermediate O2 concentrations. In case of low-KO2
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cremaster muscle network are not cuboidal and O2 concentrations were

only computed for tissue points <50 m away from any vessel
(approximately indicated by a white line). (CF) Show frequency
histograms of distances to the nearest blood vessel (C), vessel
diameters (D), blood flow rates (E), and O2 concentrations (F). The red
column in (F) represents the fraction at 00.13 M O2 (i.e., below the
KO2 value for a representative Type II HAP).
HAP this improved complementation of radiation-induced killing

(Figure 4B), but in case of high-KO2 HAP it resulted in a loss of
hypoxic selectivity at a 100-fold higher k metP,max (Figure 4E). If the
effector was allowed to diffuse through the plasma membrane (i.e.,
the bystander model), killing was increased across the whole tumor
microregion, and killing <0.13 M was less affected by decreasing
prodrug penetration with increasing k metP,max (Figures 4C,F).
Figures 4C,E illustrate the dependence on prodrug activation
rate constant for HAP of Class II (low KO2 + bystander) and Class
I (high KO2 , no bystander) respectively. It should be noted that in
the no-bystander simulations the effector potency, a, was set to a
~fivefold lower value (0.0414 M1 ) than for Class II in Table 2.
FIGURE 4 | Dependence of PK/PD in the FaDu tumor microregion on

prodrug activation rate constant k met P, max . SR-PK/PD model simulations
for HAP with KO2 = 0.13 M (AC) and a 10-fold higher KO2 of 1.3 M
(DF) are shown as a function of O2 concentration using a k met P, max of
0.001 s1 (blue), 0.01 s1 (black), 0.1 s1 (red), or 1 s1 (green).
(A,D) Intracellular prodrug AUC (AUCi P ). (B,E) Survival probability after
This was done in order to achieve similar overall (average) killing

additional to radiation in the tumor microregion for Class I and II
HAP under the default conditions (shown in black). Under these
conditions, the Class I HAP provides more killing at intermediate
O2 concentrations (~0.210 M), while the Class II HAP provides
much higher cell kill under severe hypoxia (<0.2 M O2 ).
Figures 5A,B show overall HAP-mediated killing, averaged
across the whole tumor microregion, for the cases demonstrated
in Figure 4. In all cases, monotherapy activity increased with
increasing prodrug activation rates over the range examined
(k metP,max = 0.0011 s1 ), although more markedly for low-KO2
compounds (shown in red). This difference was even more pronounced for killing of radiobiologically hypoxic cells (i.e., killing
additional to radiation; Figure 5B), in which case activity of
high-KO2 compounds (shown in green) decreases at high rates of
metabolism reflecting a severe penetration problem (which is not
fully offset by allowing bystander diffusion). Class II HAP (dark
red) are thus more tolerant of high values of k metP,max than Class
I HAP (light green).
The above analysis suggests that monotherapy activity increases
monotonically with k metP,max up to very high values, but it is
important to consider whether this changes selectivity relative
to normal tissues. To address this we used the above cremaster muscle microregion (Figure 3), to represent a generic wellperfused normal tissue with mild physiological hypoxia. For the
present purpose, we assumed that this normal tissue is populated by cells with the same intrinsic sensitivity to effector as cells
in the tumor microregion. Cell kill in the normal tissue region
showed an even steeper dependence on k metP,max than the tumor
HAP exposure or 15 Gy radiation (gray) in the absence of bystander

effects. (C,F) Survival probability with bystander effects. For no-bystander
simulations, the rate constant for membrane transfer of the effector k t E
was set to 0 and effector potency was set to a lower value of
0.0414 M1 h1 , to achieve similar average tumor cell kill as in the
bystander case. All remaining parameters were as reported in Table 2.
region (Figure 5C). As a consequence, tumor selectivity (assessed

using the tumor:normal tissue ratio of log cell kill) fell as rates
of bioreductive metabolism increased (Figure 5D). This falling
therapeutic ratio reflects the greater compromise to delivery of
rapidly metabolized prodrugs in the tumor network because of
lower blood flow rates, longer diffusion distances, and more severe
hypoxia than in the normal tissue (Figure 3). The model confirmed our expectation that toxicity to physiologically hypoxic
normal tissues will be higher for a high-KO2 HAP (Figure 5C).
MODULATION OF PRODRUG DIFFUSION PROPERTIES
We next asked whether the activity of class II HAP is sensitive to

the diffusibility of the prodrug, as previously shown by the SRPK/PD model for tirapazamine analogs (54). In the latter study,
tissue diffusion was defined by a single parameter, the effective
diffusion coefficient as measured by flux through multicellular
layer cultures; this represents the weighted averages of the free
drug diffusion coefficient within cells, across membranes and in
the extracellular matrix, based on treating tissue as homogenous
space. In contrast, the present model explicitly considers extra- and
intracellular compartments, and overall diffusion is determined
by two parameters: the rate constant for transfer between extraand intracellular compartments, k tP , and the diffusion coefficient
in the extracellular compartment D P (Figure 2). Since changes
in the physicochemical properties of HAP are expected to affect
membrane permeability more than paracellular diffusion, k tP was
modulated. The results (Figure 6) show that hypoxic cell killing for
Class I HAP is optimal at k metP,max ~0.1 s1 [Figure 6A; consistent
with the previous SR-PK/PD model for tirapazamine (54)]. Class
Table 2 | Parameters and variables of the SR-PK/PD model (base model).

Parameter
Value
Unit
Description
AUCP inflow
50.0
M/h
Unbound AUC of prodrug in inflowing vessels
k met P,max
0.01
s1
Maximum rate constant for metabolism of prodrug to effector (under anoxic conditions)
KO2
0.126 (low); 1.26 (high)
M; M
O2 concentration for half-maximum prodrug activation, assumed to be equal to the O2

concentration for half-maximum cytotoxicity of PR-104A (low KO2 ) and tirapazamine (high
KO2 ) determined in SiHa single cell suspensions (31)
0.01
s1
Rate constant for loss of effector
K tP
K tE
0.1
0.01
s1
Rate constants for transfer from the extracellular to the intracellular compartment (k tN ) for
s1
prodrug and effector
DP
DE
106
cm2 /s
Tissue diffusion coefficients for prodrug and effector. Reported values are the
106
cm2 /s
volume-averaged parameters of the extracellular diffusion coefficients DeN with: D = e DeN
0.4
k metE
Intracellular volume fraction in tumors
0.200
M1 h1
Proportionality constant for the PK/PD model for +bystander case
0.0414
M1 h1
Proportionality constant for no-bystander case
0.0663
Gy1
Proportionality constants for the linear-quadratic model for radiosensitivity under hypoxia
0.0028
Gy2
OER = OER
2.8
Kms
4.2
O2 concentration for half-maximum radiosensitivity calculated from Ref. (91)
Dr
15
Gy
Radiation dose
Maximal O2 enhancement ratios for the and components of the linear-quadratic model (91)
The parameter and variables are similar to those estimated for our previous SR-PK/PD model for PR-104 in SiHa tumors (35).
I HAP also showed an optimum value of k tP , reflecting the fact

that the membrane transfer rate constant controls how much prodrug is available for intracellular activation. In contrast, for Class
II HAP killing additional to radiation increased monotonically
over the full range of k metP,max and k tP (Figure 6B) reflecting the
tolerance of high rates of intracellular prodrug activation owing
to a low KO2 and because effector diffusion offsets compromised
prodrug penetration.
MODULATION OF THE EFFECTOR DIFFUSION RANGE
Next, we investigated the dependence of antitumor activity on

effector parameters for Class II HAP. Since tissue transport of the
effector is dependent on its membrane transfer (k tE ), paracellular diffusion (DE ) and its stability (k metE ), these parameters were
modulated ~10-fold relative to the base model. In order to see
an impact of a change in the paracellular diffusion coefficient DE ,
k tE had to be increased 10-fold to 0.1s1 , indicating that effector
transport is otherwise limited by membrane transfer. An increase
in diffusion coefficient, DE (Figure 7A) caused increased diffusion of the effector away from the hypoxic regions where it is
formed, which decreased killing in hypoxic regions and increased
killing in aerobic regions. As a consequence, overall killing in addition to radiation decreased and monotherapy activity increased
(Figures 7B,C). A similar effect could be observed when increasing the membrane transfer rate constants k tE (Figures 7D,E).
However, the effect of increased diffusibility (higher DE or k tE )
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on single-agent activity was only minor because the effector was

defined to freely permeate the blood vessel walls, and thus lost
into the vasculature. The protective effect of this washout was confirmed by simulations with zero vessel permeability to the effector,
which showed a much larger increase in HAP monotherapy activity with increasing k tE (Figures 7G,H). Notably, an increase in
effector diffusibility in the presence of vessel permeability elevated
the tumor:normal tissue ratio of killing (Figures 7C,F), reflecting
that washout of effector into the circulation is more protective in
normal tissue (with lower distances to nearest vessel; Figure 3)
than in tumor tissue.
The effect of increasing the rate constant for effector reaction
k metE was investigated using the default case where cell killing
scales with effector reactivity (case 1) and alternatively where cell
killing is independent of reactivity (case 2). The proportionality
constant a was set to the values given in Table 2 to achieve equivalent killing of the two effector types at the default k metE of 0.01 s1 .
In case 1, an increase in k metE produces a proportional increase in
the rate of formation of cytotoxic lesions and resulted in higher
killing in hypoxic regions (Figure 8A). Killing in aerobic regions
(>4 M O2 ) was decreased at high values of k metE due to associated low effector penetration, and this limited overall activity
(Figure 8B). Tumor selectivity decreased with increasing k metE
(Figure 8C) due largely to increasing cell killing in normal tissue.
In case 2 (where cytotoxic lesion production is independent
of effector reactivity) decreasing k metE resulted in higher killing
FIGURE 5 | Impact of the prodrug activation rate constant, k met P, max , on

overall cell killing in the tumor microregion of low-KO2 (red) and high-KO2
(green) HAP with and without bystander effects. Average log cell kill in the
tumor microregion by HAP without radiation (A) and additional to radiation
(B). The latter was calculated as the difference between overall log cell kill by
across the whole tumor microregion (Figure 8D), with a large

increase in HAP activity, both as a single agent and in addition to
radiation, with increasing effector stability (Figure 8E). Killing in
the normal tissue microregion was affected to a similar extent, so
that tumor selectivity was comparable for different values of k metE
(Figure 8F).
ESTIMATION OF THE IMPACT OF EFFECTOR WASHOUT BY BLOOD FLOW
The above analysis demonstrated that washout of effector by blood

flow can, under some conditions, significantly protect perivascular
cells from cytotoxicity. This raises the question whether effector washout might have pharmacodynamic effects in downstream
tumor regions (redistribution of effector through the tumor vasculature), or might result in significant systemic exposure to the
effector. To test the sensitivity of effector washout on different
parameters, we used the Class II HAP model to calculate net transport of effector across the tumor/plasma boundary in each vessel
segment of the tumor microregion. This showed high sensitivity
to the rate constant of effector production (k metP,max ) and effector
stability (k metE ) but only moderate sensitivity to effector diffusibility (k tE and DE ) (Figure 9), because the former parameters
substantially affect overall effector concentrations.
The impact on systemic exposure will depend on the distribution volume (V D ) and clearance (CL) of the effector. To evaluate
prodrug + radiation and log cell kill by radiation alone. (C) Average
HAP-mediated killing in a hypothetical normal tissue in which cells are
assumed to have the same intrinsic sensitivity as tumor cells, based on
simulations in a microvascular network of a mapped rat cremaster muscle
region. (D) Tumor:normal tissue ratio of cell killing.
a specific case, we returned to the SR-PK/PD model for PR-104

(35) because the plasma AUC of its active metabolites (PR-104H
and PR-104M) after dosing PR-104 has been determined in three
strains of mice (35, 52, 61). To facilitate this analysis, we measured
the pharmacokinetics of PR-104H and PR-104M in plasma and
liver following i.v. dosing of NIH-III nude mice with synthetic PR104H (Figure 10). This showed a very short half-life (2.6 min) for
PR-104H in plasma, and high PR-104M concentrations in plasma
and liver, indicating rapid conversion of PR-104H to PR-104M.
The half-lives of PR-104M in plasma and liver (~7 min) were similar to the half-life of PR-104M in culture medium at 37C [6.6 min
(35)], suggesting that CL of PR-104M is mainly due to its chemical
instability.
Since washout was most sensitive to k metP,max (Figure 9), we
performed PR-104 SR-PK/PD model simulations using two different values of this parameter, corresponding to rates measured
in vitro for HCT116/WT and a cell line with 20-fold higher
k metP,max (HCT116/sPOR#6) (35). This confirmed that washout
of metabolites produced in hypoxic regions can increase plasma
levels in nearby vessels, an effect that was more pronounced
at the higher k metP,max (white arrows in Figure 11). Using V D
and CL estimates from measured plasma PK following administration of PR-104 or PR-104H (Table 3), we calculated that
washout would elevate the free plasma AUC of PR-104H+M in
DISCUSSION
In this study, SR-PK/PD modeling was utilized to identify strategies for optimization of HAP. The model has a solid biological
foundation because it is based on our previous SR-PK/PD model
for PR-104 for which parameters have been determined experimentally using three cell lines (35). The latter model was the first
to explicitly consider the intra-tumor distribution of bystander
metabolites.
NORMAL TISSUE TOXICITY
FIGURE 6 | Dependence of HAP-induced cell killing additional to

radiation on k met P, max and the membrane transfer rate constant k t P .
(A) Class I HAP; (B) Class II HAP. Simulations for Class I HAP (no-bystander)
were performed as described in the legend of Figure 4.
NIH-III nude mice [estimated from the measured PK after i.p.

administration of 562 mol/kg PR-104 (35)] by only 0.8% in case
of HCT116/WT tumors, and 3.3% in case of HCT116/sPOR#6
tumors (Table 3). This was calculated assuming all PR-104H+M
was released at once and hence represents a maximum addition to
the circulating metabolites. This is consistent with published data
showing similar plasma concentrations of reduced metabolites
in NIH-III nude mice with HCT116/WT and HCT116/sPOR#6
tumors 30 min after i.p. administration of 562 mol/kg PR104 (35).
It should be noted that all simulations in this study were
performed at blood flows where prodrug delivery and effector extraction were not highly perfusion limited. To demonstrate this (and the effect of blood flow on increasing effector
washout into the systemic circulation) we increased the blood
flow rate, Q, 2.5-fold, while decreasing inflow pO2 , to maintain a similar hypoxic fraction (Figures S1A,B in Supplementary Material). Under conditions of high prodrug metabolism
(k metP,max of 1 s1 ), this increased prodrug extraction from the
plasma 1.5-fold and effector washout into the plasma twofold
(Figure S1C in Supplementary Material) but overall killing in
the microregion increased by only 26% (Figures S1D,E in Supplementary Material) reflecting little increase in perivascular effector
concentrations.
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A novel feature of the present study is the comparison of predictions for a tumor and normal tissue network, the latter based on
a mapped microvascular network in a rat cremaster muscle (56)
with oxygen consumption rate adjusted to simulate mild hypoxia
(Figure 3). This comparison is important because the utility of
HAP is likely to be constrained by the requirement that prodrug
activation at intermediate O2 does not cause toxicity in tissues
with physiological hypoxia, such as liver (4345), retina (66), and
possibly bone marrow (49). The low tumor:normal tissue ratios
of killing determined in our study (Figures 5, 7, and 8) point to
limitations on achieving tumor selectivity with HAP. However, it
is important to note that our model makes the assumption that
intrinsic cellular sensitivity to the released effector (potency parameter a) is the same for cells in the tumor and normal tissue
networks. Most effectors of HAP in clinical development are DNAreactive cytotoxins (13) that provide some tumor-specificity due to
the cytokinetics and DNA repair abnormalities (67) of tumor cells
relative to normal cells. In addition, release of HAP effectors that
target oncogenic pathways in tumor cells has potential for conferring additional tumor selectivity (68). Normal tissue models will
need to be revisited when more quantitative information about
oxygenation and drug targets in potentially dose-limiting normal
tissues is available. Nevertheless, the present results emphasize the
importance of using HAP strategies to augment selectivity of effectors that already provide some level of tumor selectivity, rather
than as the sole mechanism of tumor targeting.
In addition, normal tissue toxicity due to washout of effectors
has been a concern in the development of targeted anticancer prodrugs (69, 70) although to our knowledge no clear link between
toxicity and increased effector levels has been established. In the
current study systemic toxicity of HAP as a result of release of
active metabolites from the tumor appears unlikely; our model
suggests that a significant contribution to toxicity would require a
combination of extreme assumptions (high tumor burden, intratumor activation rates, effector stability, tumor blood flow rates, as
well as low systemic CL of effector). However, toxicity in normal
tissue sharing a microcirculatory system with the tumor remains
a possibility.
OPTIMAL PRODRUG PARAMETERS OF CLASS I AND CLASS II HAP
This study compared an idealized Class I HAP (high KO2 with nobystander effect, illustrated by tirapazamine and SN30000) and
a Class II HAP (low KO2 with bystander effect, e.g., PR-104A
and TH-302) to assess which of these HAP strategies is better
suited for complementation of radiotherapy. Our previous tirapazamine SR-PK/PD model showed that Class I HAP exhibit an
optimum k metP,max , above which hypoxic cell killing decreases
FIGURE 7 | Modulation of effector diffusion properties for a class II HAP.

SR-PK/PD model simulations using the base model (Table 2), with modulation
of the following effector parameters: (AC) the extracellular diffusion
coefficient DE , using a 10-fold higher value for the membrane transfer rate
constant k t E ; (DI) membrane transfer rate constant k t E , with (DF) or
without (GI) blood vessel permeability to the effector. Left panels show
because increasing consumption of the prodrug limits its penetration to hypoxic regions (54). This finding could be replicated using
the present model, in which intra- and extracellular compartments
are made explicit (Figure 5). In contrast, activity of Class II HAP
was predicted to increase monotonically with increasing k metP,max
in the investigated range of 0.0011 s1 ; we consider this range to
be physiologically and pharmacologically relevant based on measured rate constants (32, 35, 54, 60). In addition, the potential
to further increase k metP,max by drug design would be limited by
membrane transport and by the concurrent decrease in tumor
selectivity (Figure 5D).
The superiority of Class II over Class I HAP at high k metP,max
can be explained by two factors. Firstly, the lower KO2 value minimizes metabolic loss during diffusion into hypoxic target regions
survival probability (as a function of O2 in the FaDu tumor microregion) after

15 Gy radiation (gray) or HAP (black: base model; red and blue: 10-fold higher
and lower parameter value, respectively). Middle panels show average
monotherapy activity (black, solid lines) and killing additional to radiation
(white, dashed lines) in the tumor region. Right panels show the
tumor:normal tissue ratio of cell killing.
(compare Figures 4A,D). Secondly, bystander effects alleviate the

impact of poor prodrug penetration due to redistribution of effector between regions of different prodrug exposure. Both of these
factors also explain the lower sensitivity of antitumor activity to
prodrug diffusibility of class II relative to class I HAP (Figure 6).
DESIGN OF OPTIMIZED CLASS II HAP
Spatially resolved pharmacokinetic/pharmacodynamic modeling

represents a valuable tool for the rational design of improved HAP,
as has been demonstrated by our previous SR-PK/PD model for
Class I HAP (54). The insight that there is an optimum for combinations of DP and k metP that can accommodate the competing
requirements of efficient prodrug activation and tissue penetration
prompted the SR-PK/PD model-guided screening of tirapazamine
FIGURE 8 | Modulation of effector stability for a class II HAP. SR-PK/PD

model simulations using the base model (Table 2), or modulation of the
rate constant for loss of effector k met E . (AC) Case 1 where survival
probability is proportional to reactivity (k met E ); (DF) Case 2 where survival

probability is independent of k met E . See legend of Figure 7 for description
of graphs.
FIGURE 9 | Impact of different parameter modulations on effector

washout. Graphs show the net transport of effector across the vessel walls
in the FaDu tumor microregion, normalized to the volume of the region, as
estimated by the SR-PK/PD model with modulation of (A) k met P, max , (B) k met E ,
(C) k t E , and (D) DE , with unmodulated parameters held at their default values
from Table 2, except in (D) where 10-fold higher values for k t E was used.
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FIGURE 10 | Pharmacokinetics of PR-104H and its reduction product

PR-104M in NIH-III nude mice. Concentrations of PR-104H (filled symbols)
and PR-104M (open symbols) in plasma (A) and liver (B) following i.v.
administration of 10 mol/kg PR-104H, with linear regression lines. At each
time point different symbols represent data from individual mice.
analogs (71) that identified SN30000 as a prodrug with superior

tissue penetration and antitumor activity in combination with
radiation (32). The model-based design of class II HAP presents
a greater challenge as it requires the specific consideration of the
transport parameters of the effector in addition to those of the
prodrug. In this study SR-PK/PD modeling was used to identify
reaction-diffusion parameters of Class II HAPs that could be optimized. The outcome is summarized in Table 4, which shows that
antitumor activity of class II HAP could be enhanced by increasing k metP,max , although this would need to be balanced against the
need to maintain sufficient tumor selectivity.
When looking at the impact of a change in effector metabolism (k metE ) we distinguished two types of effectors: an effector
that needs to react irreversibly with its target to elicit cytotoxicity,
meaning that cell killing is a function of this chemical reactivity
as for alkylating agents (Case 1), and an inhibitor where chemical transformation of the effector (whether spontaneously or by
metabolism) gives rise to non-toxic products (Case 2). Modeling
showed that effector reactivity should be increased in Case I but
decreased in Case 2, in order to improve antitumor activity. In
Case 1 the increase in overall killing when increasing reactivity by
drug design needs to be balanced against a predicted decrease in

tumor selectivity.
The model revealed that bystander effects are limited not only
by diffusion and reaction of the effector, but also by effector
washout, unless this washout targets downstream regions via a
blood-borne bystander effect. The blood vessels act as a sink for
effector, a problem that has previously been flagged in the context of drug diffusion into peritoneal tumors from the peritoneal
cavity (72). Therefore, the choice of optimal effector diffusibility (defined by k tE and DE ) depends on the therapeutic context,
with low diffusibility generally better for combination with radiation, and high diffusibility preferred for monotherapy activity
(Figures 7B,E).
Notably, a greater increase of bystander killing with increasing
effector diffusibility has previously been suggested by the finding that bystander effects of dinitrobenzamide mustard prodrugs
in multicellular layer co-cultures of NfsB-overexpressing and WT
cells correlated with effector lipophilicity (73, 74). This may partially be due to the intimate mixture of activator and target cells
in these co-cultures, so that effector washout at the multicellular
layer boundaries affects both cell types to the same extent. In contrast, blood vessels in the virtual tumor microregion that act as a
sink for effector are generally more distant from hypoxic prodrugactivating regions than from bystander target zones. Therefore,
multicellular layer co-cultures have may limited use in assessing
antitumor activity for HAP, although they are useful to rank HAP
according to the diffusion range of their effectors.
Although an increase in the effector diffusion range provided
only a moderate increase in single-agent activity it was found to
have a surprisingly large effect in protecting cells in normal tissues
where the HAP is activated (Figures 7C,F). Increasing effector
diffusibility and prodrug activation rate simultaneously (the latter
to compensate for washout) may therefore be a good strategy to
improve monotherapy activity.
The suggested HAP model parameters could be modulated
in drug design by changing the physicochemical properties that
determined these parameters; e.g., one-electron reduction potential in controlling k metP,max (9, 75, 76) or lipophilicity, pKa , molecular weight, and number of H-bond donors and acceptors in
controlling diffusibility (77). In most cases any change will affect
several model parameters, but the value of the SR-PK/PD model
lies in part in its ability to accommodate all such changes.
The high prodrug diffusibility and low effector diffusibility that
would be ideal for combination with radiotherapy according to the
model predictions may not be achievable in all chemical classes of
HAP. In case of dinitrobenzamide mustards (PR-104 analogs), the
physicochemical properties of prodrug and effector (other than
mustard reactivity) will be similar since their structures only differ in one aromatic substituent. The use of fragmenting prodrugs
of aliphatic mustards such as TH-302 relaxes this constraint, providing a higher scope for independent optimization of prodrug
and effector diffusion properties.
LIMITATIONS OF THE MODEL
The present SR-PK/PD model has two key limitations. Firstly,

the model uses a small tumor region (0.12 mm3 ) that cannot be
expected to fully capture the heterogeneity in vascular density and
FIGURE 11 | Simulation of PR-104M, the second active metabolite of

PR-104A, in the tumor microregion. PR-104M AUC was calculated in the
FaDu tumor microregion with oxygenation as shown in Figures 3A,F, using
our previously published PR-104 SR-PK/PD model (35) at a PR-104 dose of
562 mol/kg with inflow AUC of PR-104H and PR-104M set to 0 to
distinguish the impact of reduced metabolites formed in the tumor.
perfusion in tumors (78). This heterogeneity results in macroregional variations in oxygenation and prodrug delivery that may
decrease the overall relevance of bystander effects of Class II
HAP. For example, macroscopic, well-perfused, and oxygenated
areas [observed in some tumor xenografts (35, 79, 80)] would be
beyond the reach of the hypoxia-driven bystander effects studied
here, unless there is redistribution of diffusible effectors between
differently oxygenated tumor regions via the bloodstream (bloodborne bystander effect). Mapped tumor regions of a larger scale
are not yet available but could be used to investigate these macroregional effects in the future. Secondly, our steady-state model does
not account for time-dependent processes such as cycling hypoxia
(8183) and the kinetics of reoxygenation, cell proliferation, and
cell death. Cycling hypoxia is thought to arise from variations in
blood flow (8486), which would affect not only delivery of oxygen but also that of the HAP. Since cycling hypoxia has a dominant
periodicity of two to three cycles per hour (82), it may be relevant even for HAP with a residence time in the circulation of few
hours and critically important for HAP with long residence times
in tumors as is reoxygenation for effectors with long residence
times such as AQ4N (87). The implications of variable blood flow
and cycling hypoxia should be incorporated in future SR-PK/PD
models.
CONCLUSION
In spite of its limitations, the SR-PK/PD model has identified
strategies for optimization of HAP worthy of further investigation. This is the first modeling study that attempts to systematically
compare the activity of Class I and Class II HAP and the first that
integrates the use of a normal tissue network to assess tumor selectivity. It suggests that class II HAP offer the following advantages
relative to class I HAP: they tolerate a wider range of k metP,max
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(A) Simulation for an HCT116/WT tumor. (B) Simulation for a

HCT116/sPOR#6 tumor (20 higher prodrug activation rate constant). The
AUC (in micromoles hour) is shown in color scale in the whole FaDu
microvascular network projected into one plane, and in a mid-plane section
of the microregion (as a contour plot). White arrows indicate vessels with
high concentrations of active metabolite.
(allowing a wider scope for prodrug design as well as their use

in a range of human tumors with different one-electron reductase
expression), are less sensitive to prodrug diffusibility (thus making
it less critical to optimize this parameter) and have lower toxicity in
normal tissues with physiological hypoxia (Figures 4C,F). A possible disadvantage is that complementation of radiation falls off
rapidly with increasing effector diffusion away from the hypoxic
activating region (Figure 7).
The modeling also showed that the design of optimized Class
II HAP is complex due to the need to consider many different
processes such as metabolism, membrane transfer, paracellular
diffusion, and washout. The spatial and temporal heterogeneity
of O2 and blood flow within tumors (and potentially variations in
reductase expression and other determinants of sensitivity) adds
further complexity. Therefore it is difficult to design one HAP that
fits all tumor types and therapeutic settings. However, important
tendencies have been identified by the model, e.g., that antitumor
activity may be increased by optimizing effector stability and prodrug activation rates while tumor selectivity may be improved by
increasing effector diffusibility.
The present study has implications for targeted anticancer
prodrug approaches, as most are limited by spatial heterogeneity in prodrug activation (3, 4, 88). The approach is also
potentially applicable to hypoxia-targeting strategies that utilize prodrugs such as gene-directed enzyme prodrug therapy
approaches in which the prodrug-activating enzyme is delivered
using macrophages that accumulate in hypoxic tumor regions
(89), or obligate anaerobic bacteria (90). In approaches with a different spatial distribution of prodrug-activating regions relative to
non-activating regions and blood vessels the optimum conditions
may differ and could be identified in the future using SR-PK/PD
modeling.
Table 3 | SR-PK/PD model estimation of the free AUC of PR-104A,
AUTHOR CONTRIBUTIONS
PR-104H, and PR-104M extracted from or released into plasma by a
Tumor
PR-104A PR-104H PR-104M
Net amount extracted
WT
80.2
3.37
11.77
()/released (+) in the tumor
sPOR#6 300
14.4
48.7
Net amount extracted
WT
2.02
7.06
()/released (+) by a
sPOR#6 180
8.61
29.2
Annika Foehrenbacher, William R. Wilson, and Kevin O. Hicks

conceived and designed the study. Timothy W. Secomb developed
the Greens function method and wrote the program to simulate
multiple intracellular and extracellular solutes. Annika Foehrenbacher designed and ran the simulations and Annika Foehrenbacher, Kevin O. Hicks, and William R. Wilson analyzed the data.
Annika Foehrenbacher performed the PR-104H pharmacokinetic
study and assembled the figures, table, and manuscript. Annika
Foehrenbacher, Kevin O. Hicks, William R. Wilson, and Timothy
W. Secomb wrote the paper.
2.6a
2.8b
2.8b,c
ACKNOWLEDGMENTS
5.0d
44b
20b,c
0.286
0.00179
0.0140
0.00759
0.0578
HCT116 tumor following i.p. administration of 562 mol/kg PR-104.
microregion (pmol/mm3 )
48.1
600 mm3 tumor in 1 h (nmol)

V D (l/kg)
CL (l/h/kg)
Free AUC extracted
WT
()/released (+) by a
sPOR#6 1.07
This work was supported by the Health Research Council of New

Zealand (Grant number 11-1103) and a University of Auckland
International Doctoral Scholarship to Annika Foehrenbacher.
SUPPLEMENTARY MATERIAL
600 mm3 tumor (M.h)

Free AUC in plasma (M.h)c
61.5
1.30
0.71
Using our previous PR-104 SR-PK/PD model for HCT116/WT tumors and
The Supplementary Material for this article can be found

online at http://www.frontiersin.org/Journal/10.3389/fonc.2013.
00314/abstract
HCT116/sPOR#6 tumors [with a 20-fold higher prodrug activation rate constant

(35)], simulations were performed for a PR-104 dose of 562 mol/kg, with inflow
AUC of PR-104H and PR-104M from extra-tumor sources set to 0 to identify the
flux of reduced metabolites formed in the tumor. Net transport of PR-104A, PR104H, and PR-104M across the tumor-plasma boundary in the tumor microregion
was calculated, scaled to the volume of a typical HCT116 tumor (600 mm3 ), and
divided by the clearance (CL) for a 25 g mouse to calculate the AUC reached in
plasma. The distribution volume (VD ) and CL were determined in pharmacokinetic
studies (see footnotes).
a
Estimated for CD-1 mice following i.v. administration of 56.2 mol/kg PR-104A
(92).
b
Estimated from the plasma PK of PR-104H and PR-104M measured in NIH-III
Figure S1 | Impact of blood flow rate on tumor PK/PD of HAP. SR-PK/PD

model simulations were performed using the default O2 transport parameters in
Table 1 (Q = 40 nl/min; pO2 = 40 mm Hg; black) and using a higher blood flow
rate and lower inflow pO2 (Q = 100 nl/min; pO2 = 29 mm Hg; red). The prodrug
activation rate constant k met P, max was set to a high value of 1 s1 with all other
parameters as in Table 2. (A) O2 concentration as a function of distance to
nearest vessel in the FaDu tumor microregion. (B) The resulting oxygen
distribution for the higher blood flow rate case (c.f. Figure 3F). (C) Net amount
of prodrug and effector extracted from plasma ( sign) or released into plasma
(+ sign). (D) Intracellular prodrug concentration CiP as a function of O2 in the
FaDu tumor microregion. (E) Resulting killing. Lines indicate average killing in
the tumor region (2.47 and 3.04 logs of cell kill for low flow and high flow
simulations respectively).
nude mice after i.v. administration of 10 mol/kg PR-104H (Figure 10).

c
Assuming that VD of PR-104M is equal to VD of PR-104H.
Estimated by non-compartmental analysis of the PR-104A plasma PK measured
REFERENCES
in NIH-III nude mice after i.p. administration of 562 mol/kg PR-104 (35).
Table 4 | Summary of SR-PK/PD modeling results for Class II HAP.

Antitumor
Tumor
Systemic effector
activitya
selectivityb
releasec
HAP
HAP+
only
radiation
k metP,max
k metE (case 1)
k metE (case 2)
DE
k tE
increase; decrease; ~ no substantial change.

a
Overall cell kill predicted in the 3D FaDu tumor microregion (see Figures 5 and 7).
Ratio of overall cell killing in the tumor and cremaster muscle microregions (see
Figures 5 and 7).

c
Overall effector released into the circulation by the FaDu tumor microregion (see
Figure 8).
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 06 November 2013; paper pending published: 26 November 2013; accepted:
11 December 2013; published online: 27 December 2013.
www.frontiersin.org
Citation: Foehrenbacher A, Secomb TW, Wilson WR and Hicks KO (2013) Design

of optimized hypoxia-activated prodrugs using pharmacokinetic/pharmacodynamic
modeling. Front. Oncol. 3:314. doi: 10.3389/fonc.2013.00314
This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the
journal Frontiers in Oncology.
Copyright 2013 Foehrenbacher, Secomb, Wilson and Hicks. This is an open-access
article distributed under the terms of the Creative Commons Attribution License (CC
BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
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HYPOTHESIS AND THEORY ARTICLE

published: 27 December 2013

Design of optimized hypoxia-activated prodrugs using

Hypoxia contributes to resistance of tumors to some cytotoxic drugs and to radiotherapy,

between the reaction/diffusion properties of HAP in the tumor

December 2013 | Volume 3 | Article 314 | 1

FIGURE 1 | Hypoxia-activated prodrug strategies. (A) Schematic

and activation of TH-302 also appears to require severe hypoxia

Frontiers in Oncology | Pharmacology of Anti-Cancer Drugs

Model-based optimization of hypoxia-activated prodrugs

FIGURE 2 | Schematic representation of a generalized HAP PK/PD

HCT116 and SiHa xenografts is due to bystander effects both as

December 2013 | Volume 3 | Article 314 | 2

Model-based optimization of hypoxia-activated prodrugs

medium) was calculated based on previous estimates for blood

production equal to the rate of loss of prodrug: rE = i k metP C iP .

Inflow of prodrug to the microvascular networks was defined by its

Survival probability (SP) at each point of the tumor microregion

Volume of the mapped region

O2 partial pressure in inflowing vessels

O2 partial pressure for half saturation of hemoglobin

Hill coefficient for O2 binding to hemoglobin

O2 binding capacity of red blood cells

cm3 O2 /cm3 /mm Hg

Rate of O2 consumption when supply is not limiting

Michaelis constant for O2 consumption

December 2013 | Volume 3 | Article 314 | 3

Model-based optimization of hypoxia-activated prodrugs

where the SP is averaged over the whole tumor microregion.

The SR-PK/PD model calculates the transport of prodrug and

PR-104H (33) and PR-104M (61) were prepared from PR-104A

Frontiers in Oncology | Pharmacology of Anti-Cancer Drugs

We used a digitized 3D tumor microregion (Figure 3A) that was

December 2013 | Volume 3 | Article 314 | 4

FIGURE 3 | Virtual tissue microregions used for SR-PK/PD modeling.

prodrug activation rate constant for anoxia (k metP,max ) under these

Model-based optimization of hypoxia-activated prodrugs

cremaster muscle network are not cuboidal and O2 concentrations were

HAP this improved complementation of radiation-induced killing

December 2013 | Volume 3 | Article 314 | 5

FIGURE 4 | Dependence of PK/PD in the FaDu tumor microregion on

This was done in order to achieve similar overall (average) killing

Frontiers in Oncology | Pharmacology of Anti-Cancer Drugs

Model-based optimization of hypoxia-activated prodrugs

HAP exposure or 15 Gy radiation (gray) in the absence of bystander

region (Figure 5C). As a consequence, tumor selectivity (assessed

We next asked whether the activity of class II HAP is sensitive to

December 2013 | Volume 3 | Article 314 | 6

Model-based optimization of hypoxia-activated prodrugs

Table 2 | Parameters and variables of the SR-PK/PD model (base model).

Unbound AUC of prodrug in inflowing vessels

0.126 (low); 1.26 (high)

O2 concentration for half-maximum prodrug activation, assumed to be equal to the O2

Rate constant for loss of effector

prodrug and effector

volume-averaged parameters of the extracellular diffusion coefficients DeN with: D = e DeN

Intracellular volume fraction in tumors

Proportionality constant for the PK/PD model for +bystander case

Proportionality constant for no-bystander case

O2 concentration for half-maximum radiosensitivity calculated from Ref. (91)

I HAP also showed an optimum value of k tP , reflecting the fact

Next, we investigated the dependence of antitumor activity on

on single-agent activity was only minor because the effector was